U.S. patent application number 12/861521 was filed with the patent office on 2010-12-16 for methods, particles, and kits for determining activity of a kinase.
This patent application is currently assigned to LUMINEX CORPORATION. Invention is credited to Michaela R. Hoffmeyer, James W. Jacobson, Ananda G. Lugade.
Application Number | 20100317544 12/861521 |
Document ID | / |
Family ID | 38541967 |
Filed Date | 2010-12-16 |
United States Patent
Application |
20100317544 |
Kind Code |
A1 |
Jacobson; James W. ; et
al. |
December 16, 2010 |
Methods, Particles, and Kits for Determining Activity of a
Kinase
Abstract
Methods, particles and kits for determining kinase activity
within a sample are provided. An embodiment of a method includes
exposing a fluorescent particle to an assay, wherein the
fluorescent particle includes a support substrate having one or
more fluorescent materials and a peptide substrate coupled to the
support substrate via a functional group of the support substrate.
The method further includes phosphorylating the peptide substrate
during exposure of the fluorescent particle to the assay and
processing the fluorescent particle such that the peptide substrate
is dephosphorylated and a polarized double bond is generated at a
dephosphorylated site. In addition, the method includes coupling a
fluorescent reporter having a nucleophilic terminal group to the
fluorescent particle via the polarized double bond.
Inventors: |
Jacobson; James W.;
(Leander, TX) ; Lugade; Ananda G.; (Austin,
TX) ; Hoffmeyer; Michaela R.; (Cedar Park,
TX) |
Correspondence
Address: |
DAFFER MCDANIEL LLP
P.O. BOX 684908
AUSTIN
TX
78768
US
|
Assignee: |
LUMINEX CORPORATION
Austin
TX
|
Family ID: |
38541967 |
Appl. No.: |
12/861521 |
Filed: |
August 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11736254 |
Apr 17, 2007 |
7795040 |
|
|
12861521 |
|
|
|
|
60744949 |
Apr 17, 2006 |
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Current U.S.
Class: |
506/11 ; 435/15;
506/18 |
Current CPC
Class: |
C12Q 1/485 20130101 |
Class at
Publication: |
506/11 ; 435/15;
506/18 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48; C40B 30/08 20060101 C40B030/08; C40B 40/10 20060101
C40B040/10 |
Claims
1. A method, comprising: exposing a fluorescent particle to an
assay, wherein the fluorescent particle comprises: a support
substrate comprising one or more fluorescent materials; and a
peptide substrate coupled to the support substrate via a functional
group of the support substrate; phosphorylating the peptide
substrate during the step of exposing the fluorescent particle to
the assay; processing the fluorescent particle such that the
peptide substrate is dephosphorylated and a polarized double bond
is generated at a dephosphorylated site; and coupling a fluorescent
reporter comprising a nucleophilic terminal group to the
fluorescent particle via the polarized double bond, wherein the
fluorescent reporter is configured to emit fluorescence within a
different wavelength range than the one or more fluorescent
materials of the support substrate.
2. The method of claim 1, wherein the fluorescent reporter
comprises: a fluorescent compound; and one or more spacer compounds
interposed between the nucleophilic terminal group and the
fluorescent compound.
3. The method of claim 2, wherein the one or more spacer compounds
collectively comprise between approximately 1 atom and
approximately 25 atoms.
4. The method of claim 1, wherein the fluorescent reporter is
configured to emit fluorescence at a wavelength greater than
approximately 500 nm.
5. The method of claim 1, wherein the step of phosphorylating the
peptide substrate comprises exposing the fluorescent particle to an
ionic liquid heated via microwaves.
6. The method of claim 1, wherein the step of processing the
fluorescent particle comprises exposing the fluorescent particle to
an ionic liquid.
7. The method of claim 6, wherein the step of processing the
fluorescent particle further comprises microwave heating the
fluorescent particle and the ionic liquid.
8. The method of claim 1, wherein the step of coupling the
fluorescent reporter comprises exposing the fluorescent particle to
an ionic liquid.
9. The method of claim 8, wherein the step of coupling the
fluorescent reporter further comprises microwave heating the
fluorescent particle and the ionic liquid.
10. A method, comprising: exposing a pooled population of different
subsets of fluorescent particles to a sample, wherein at least some
of the fluorescent particles comprise: a support substrate
comprising one or more fluorescent materials configured to emit
fluorescence in a first wavelength range, and wherein at least some
of the different subsets of fluorescent particles respectively
comprise a different configuration of the one or more fluorescent
materials; and a peptide substrate coupled to the support substrate
via a functional group of the support substrate, and wherein at
least some of the different subsets of fluorescent particles
respectively comprise a different peptide substrate; exposing the
sample and the pooled population to a phosphorylation process
configured to add phosphate groups to accepting residues of the
peptide substrates; processing a plurality of the fluorescent
particles subsequent to exposing the sample and the pooled
population to the phosphorylation process such that if any
phosphorylated peptide substrates exist among the plurality of
fluorescent particles, the phosphorylated peptide substrates are
dephosphorylated and polarized double bonds are generated at
dephosphorylated sites of the peptide substrates; further
processing the plurality of the fluorescent particles such that if
any polarized double bonds exist among the dephosphorylated sites
of the peptide substrates, fluorescent reporters are coupled to the
fluorescent particles at positions of the polarized double bonds
via nucleophilic terminal groups of the fluorescent reporters,
wherein the fluorescent reporters are configured to emit
fluorescence in a second wavelength range distinct from the first
wavelength range; measuring fluorescence emissions of the plurality
of the fluorescent particles subsequent to further processing the
plurality of the fluorescent particles; identifying subset
classifications of the particles in the sample based upon measured
fluorescence emissions within the first wavelength range; and
determining, based upon the existence of or lack of measured
fluorescence emissions within the second wavelength range, an
amount of kinase activity within the sample when the sample and the
pooled population are exposed to the phosphorylation process.
11. The method of claim 10, wherein the step of identifying subset
classifications of the particles comprises determining the identity
of more than approximately 100 subset classifications.
12. The method of claim 10, wherein the step of further processing
the plurality of the fluorescent particles comprises: coupling a
first fluorescent reporter to fluorescent particles within a first
subset of the plurality of fluorescent particles; and coupling a
different fluorescent reporter to fluorescent particles within a
second subset of the plurality of fluorescent particles, and
wherein the different fluorescent reporter is configured to emit
fluorescence in a wavelength range distinct from a wavelength range
the first fluorescent reporter is configured to emit.
13. A kit, comprising: a plurality of fluorescent particles,
wherein each of the plurality of fluorescent particles comprises a
support substrate with one or more fluorescent materials configured
to emit fluorescence in a first wavelength range; and a plurality
of kinase-specific peptide substrates respectively coupled to
different subsets of the plurality of fluorescent particles via
functional groups of the support substrates.
14. The kit of claim 13, further comprising: a phosphorylation
reagent configured to phosphorylate the kinase-specific peptide
substrates; and a beta-elimination reagent configured to
dephosphorylate the kinase-specific peptide substrates and generate
Michael acceptors at the dephosphorylation sites of the
kinase-specific peptide substrates.
15. The kit of claim 14, wherein at least one of the
phosphorylation reagent and the beta-elimination reagent comprises
an ionic liquid.
16. The kit of claim 13, further comprising one or more fluorescent
reporter reagents each having a nucleophilic terminal group and one
or more fluorescent compounds configured to emit fluorescence in a
wavelength range distinct from the first wavelength range.
17. The kit of claim 16, wherein the one or more fluorescent report
reagents comprise a plurality of fluorescent report reagents which
are respectively configured to emit fluorescence in a different
wavelength range.
18. The kit of claim 16, wherein at least one of the one or more
fluorescent reporter reagents comprises a hydrophilic fluorescent
compound.
19. The kit of claim 16, wherein at least one of the one or more
fluorescent reporter reagents comprises an ionic liquid.
Description
PRIORITY CLAIM
[0001] The present application is a divisional from U.S. patent
application Ser. No. 11/736,254 filed Apr. 17, 2007 which claims
priority to U.S. Provisional Application No. 60/744,949 filed Apr.
17, 2006.
BACKGROUND OF THE INVENTION
[0002] 1. Field Of The Invention
[0003] The present invention generally relates to methods and
compositions (e.g., particles and kits) for determining activity of
a kinase or kinases and, more specifically, to methods and
compositions for determining activity/activities of one or more
kinases coupled to particles in a multiplexing process using
fluorescence detection.
[0004] 2. Description of the Related Art
[0005] The following description and examples are not admitted to
be prior art by virtue of their inclusion in this section.
[0006] Protein kinases play an important role in regulating
cellular signal transduction within living organisms and readily
occur in nature. For example, there are more than 500 protein
kinases and over 500,000 human phosphorylation sites in the human
genome. A protein kinase can be generally defined as an enzyme
catalyzing the transfer of phosphate from adenosine triphosphate
(ATP) to an amino acid residue. Abnormal expressions of protein
phosphorylation events may be associated with several diseases and
malignancies in living organisms, particularly humans. As such,
monitoring protein kinase activity may be advantageous for
detecting diseases and malignancies and/or identifying therapeutic
agents for diseases and malignancies (i.e., therapeutic agents for
promoting or inhibiting protein kinase activity within a living
organism).
[0007] As apparent to one skilled in the art of microarray
technology, it is generally advantageous to determine the presence
and/or concentration of analytes within chemical and biological
assays quickly. In addition or alternatively, it may be
advantageous to evaluate multiple analytes simultaneously. The
simultaneous evaluation of multiple analytes within a single sample
is referred to herein as a multiplexing scheme. Conventional
techniques for determining kinase activity are not typically
suitable for high throughput screening and/or a multiplexing assay.
In particular, many conventional techniques for determining kinase
activity utilize radioactive isotopes and rely on liquid
chromatography and/or mass spectrometry for analysis and,
therefore, are not suitable for rapid examination. In addition,
such methods do not continuously monitor kinase activity and,
consequently, may not render an accurate determination of kinase
activity. Other techniques for determining kinase activity involve
expensive and specialized biological reagents such as
phosphopeptide-specific antibodies. In general, antibody-based
microarrays produce a large number of false positives and negatives
due to the unpredictable cross-reactivity of antibodies.
Consequently, antibody-based kinase activity techniques are
generally not amenable to high throughput screening and/or
multiplexing assays. Other approaches for determining kinase
activity utilize fluorescent sensors which undergo a conformational
change upon phosphorylation. A majority of fluorescent sensors
employed in conventional assays, however, demonstrate very modest
fluorescence changes on phosphorylation, which limits their
applicability.
[0008] Accordingly, it would be advantageous to develop new
methods, particles, and kits for determining kinase activity within
an assay, particularly ones that are suitable for high throughput
screening and/or multiplexing.
SUMMARY OF THE INVENTION
[0009] The following description of various embodiments of methods,
particles, and kits for determining kinase activity is not to be
construed in any way as limiting the subject matter of the appended
claims.
[0010] An embodiment of a method for processing a particle includes
exposing a fluorescent particle to an assay, wherein the
fluorescent particle includes a support substrate having one or
more fluorescent materials and a peptide substrate coupled to the
support substrate via a functional group of the support substrate.
The method further includes phosphorylating the peptide substrate
during exposure of the fluorescent particle to the assay and
processing the fluorescent particle such that the peptide substrate
is dephosphorylated and a polarized carbon to carbon double bond
(hereinafter referred to as a polarized double bond) is generated
at a dephosphorylated site. In addition, the method includes
coupling a fluorescent reporter having a nucleophilic terminal
group to the fluorescent particle via the polarized double
bond.
[0011] An embodiment of a particle includes a support substrate
having one or more fluorescent materials and a peptide substrate
coupled to the support substrate via a functional group of the
support substrate.
[0012] An embodiment of a kit for detecting an amount of kinase
activity within a sample includes a plurality of fluorescent
particles and one or more kinase-specific peptide substrates. Each
of the plurality of fluorescent particles includes a support
substrate with one or more fluorescent materials configured to emit
fluorescence in a first wavelength range.
[0013] An embodiment of a method for detecting an amount of kinase
activity within a sample includes exposing a pooled population of
different subsets of fluorescent particles to the sample. At least
some of the fluorescent particles include a support substrate with
one or more fluorescent materials configured to emit fluorescence
in a first wavelength range, wherein at least some of the different
subsets of fluorescent particles respectively include a different
configuration of the one or more fluorescent materials. In
addition, at least some of the fluorescent particles include a
peptide substrate coupled to the support substrate via a functional
group of the support substrate, wherein at least some of the
different subsets of fluorescent particles respectively include a
different peptide substrate. The method further includes exposing
the sample and the pooled population to a phosphorylation process
configured to add phosphate groups to accepting residues of the
peptide substrates.
[0014] Furthermore, the method includes subsequently processing a
plurality of the fluorescent particles such that if any
phosphorylated peptide substrates exist among the plurality of
fluorescent particles, the phosphorylated peptide substrates are
dephosphorylated and polarized double bonds are generated at
dephosphorylated sites of the peptide substrates. Moreover, the
method includes further processing the plurality of the fluorescent
particles such that if any polarized double bonds exist among the
dephosphorylated sites of the peptide substrates, fluorescent
reporters are coupled to the fluorescent particles at positions of
the polarized double bonds via nucleophilic terminal groups of the
fluorescent reporters. Such fluorescent reporters are configured to
emit fluorescence in a second wavelength range distinct from the
first wavelength range. The method also includes subsequently
measuring fluorescence emissions of the plurality of the
fluorescent particles and identifying subset classifications of the
particles in the sample based upon measured fluorescence emissions
within the first wavelength range. In addition, the method includes
determining, based upon the existence of or lack of measured
fluorescence emissions within the second wavelength range, an
amount of kinase activity within the sample when the sample and the
pooled population are exposed to the phosphorylation process.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Further advantages of the present invention may become
apparent to those skilled in the art with the benefit of the
following detailed description of the preferred embodiments and
upon reference to the accompanying drawings in which:
[0016] FIG. 1 depicts a flowchart of a method for processing a
fluorescent particle for kinase detection; and
[0017] FIG. 2 depicts a flowchart of a method for determining
kinase activity within a sample using a multiplexing assay
scheme.
[0018] While the invention is susceptible to various modifications
and alternative forms, specific embodiments thereof are shown by
way of example in the drawings and may herein be described in
detail. The drawings may not be to scale. It should be understood,
however, that the drawings and detailed description thereto are not
intended to limit the invention to the particular form disclosed,
but on the contrary, the intention is to cover all modifications,
equivalents and alternatives falling within the spirit and scope of
the present invention as defined by the appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] In general, the term "particle" as used herein may refer to
any substrate used for the analysis of chemistry and biological
assays and may specifically refer to articles used to provide
and/or support molecular reactions for the qualification and/or
quantification of an analyte of interest including but not limited
to kinase activity. In addition, the term "particle" may reference
articles of a broad range of sizes, such as but not limited to
articles having dimensions between approximately 1 nm approximately
300 .mu.m. Hence, the term "particle" may refer to a number of
different materials and configurations, including but not limited
to microspheres, beads, polystyrene beads, microparticles, gold
nanoparticles, quantum dots, nanodots, nanoparticles, composite
particles (e.g., metal-polymeric particles or magnetite-polymeric
particles), nanoshells, nanorods, nanotubes, microbeads, latex
particles, latex beads, fluorescent beads, fluorescent particles,
colored particles, colored beads, tissue, cells, micro-organisms,
spores, organic matter, any non-organic matter, or any combination
thereof. Accordingly, any of such terms may be interchangeable with
the term "particle" used herein.
[0020] Turning to the drawings, a flowchart of a method for
processing a fluorescent particle for kinase detection is shown in
FIG. 1. As shown in FIG. 1, the method may include block 10 in
which a fluorescent particle is exposed to an assay. The assay may
be a biological assay or a chemical assay. Block 10 specifies the
fluorescent particle include a support substrate having one or more
fluorescent materials and a peptide substrate coupled to the
support substrate via a functional group of the support substrate.
As noted above, the term "particle" as used herein may refer to any
substrate used for the analysis of chemistry and biological assays
and, as such, the term "fluorescent particle" may include any of
such substrates comprising one or more photoluminescent materials
(e.g., fluorophores, fluorescent dyes, or other fluorescent
materials). Although embodiments are described herein with respect
to fluorescent dyes, it is to be understood that the embodiments
described herein may be used with any photoluminescent material
(e.g., a fluorophore or a quantum dot). The photoluminescent
materials may be incorporated into the support substrates and/or
may be coupled to a surface of the support substrates. In some
embodiments, it may be particularly advantageous to crosslink the
support substrate in order to incorporate multiple photoluminescent
materials within the support substrate. Such embodiments may be
particularly applicable for multiplexing assays such that particle
classifications on the order of 100 or more may be obtained. The
support substrate of the fluorescent particle may include any of
those used for the analysis of chemistry and biological assays,
including but not limited to polystyrene, metal, or a composite of
core and shell materials.
[0021] As noted above, in addition to the support substrate of the
fluorescent particle having one or more fluorescent materials, the
fluorescent particle includes a peptide substrate coupled to the
support substrate via a functional group (e.g., COOH, NH.sub.2, OH,
etc.) of the support substrate. More specifically, the fluorescent
particle may include a kinase-specific peptide substrate coupled to
the support substrate and, in some cases, coupled to the support
substrate via a covalent bond. In this manner, the peptide
substrate may be susceptible to phosphorylation as noted in block
12 of the flowchart depicted in FIG. 1. In particular, block 12 in
FIG. 1 denotes the method including phosphorylating the peptide
substrate during the step of exposing the fluorescent particle to
the assay. Such a phosphorylation process may be conducted in the
presence of ATP as well as a kinase. The kinase may be readily
available in the sample or may be added for the phosphorylation
process. The determinants of specificity for kinases are not well
understood, although it is understood that both the amino acid
sequence motif surrounding the serine/threonine/tyrosine residues
of the peptide substrate and the three-dimensional structure of the
substrate contribute to the specificity.
[0022] In any case, the phosphorylation process may be observed on
serine, threonine or tyrosine residues of the peptide substrate. In
some embodiments, the phosphorylation process may be conducted in
the presence of an ionic liquid. It is theorized that ionic liquids
may enhance the rate of phosphorylation, decreasing the processing
time of the method, and/or reduce the generation of byproducts
which may hinder the subsequent processing of the fluorescent
particle, particularly those processes described below in reference
to blocks 14 and 16. In some cases, the ionic liquid used to
enhance the phosphorylation process may be heated via microwaves.
In general, microwave heating may further enhance the rate of
phosphorylation. More specifically, utilizing an ionic liquid
heated via microwaves may reduce the time needed to complete the
phosphorylation process to several minutes rather than several
hours.
[0023] Subsequent to phosphorylating the peptide substrate, the
fluorescent particle may be removed from the sample such that the
fluorescent particle may be subsequently processed for the
determination of kinase activity as described in more detail below.
In some embodiments, the support substrates of the fluorescent
particles may be magnetic and the removal process may involve the
application of a magnetic field to immobilize the particles while
the supernatant is removed. Such an embodiment may advantageously
simplify the removal of the particles from the sample and possibly
avoid time consuming filtration steps. In yet other cases, however,
the support substrates of the fluorescent particles may not be
magnetic and the particles may be removed via filtration.
[0024] Continuing to block 14 in FIG. 1, the method includes
processing the fluorescent particle such that the peptide substrate
is dephosphorylated and a polarized double bond is generated at a
dephosphorylated site of the peptide substrate. The
dephosphorylation process may be conducted by a base catalyzed beta
elimination process. In general, the beta elimination process may
include any base sufficient to catalyze the process. Examples of
bases include but are not limited to sodium hydroxide and
tetramethyl ammonium hydroxide (TMA). In some cases, it may be
advantageous to employ milder bases (i.e., bases having a pH less
than 14), such as TMA, to reduce the amount of byproducts produced
from the process. In some embodiments, an ionic liquid may be
combined with a milder base to further lessen the generation of
byproducts. In yet further embodiments, the base and ionic liquid
may be heated via microwaves to increase the rate of the beta
elimination reaction. In any case, the beta elimination process is
configured to remove the phosphate group from the peptide substrate
and replace it with a polarized double bond. The polarized double
bond may be referred to as a Michael acceptor, which may used in a
subsequent Michael-type addition reaction as described in more
detail below in reference to block 16.
[0025] As shown in FIG. 1, the method includes block 16 in which a
fluorescent reporter having a nucleophilic terminal group is
coupled to the fluorescent particle via the polarized double bond.
The reaction may generally be referred to as a Michael-type
reaction, which may be generally defined as addition of a
nucleophilic atom to a compound containing a polarized double bond.
The compound containing the nucleophilic atom is commonly called
the "Michael donor," and the compound containing the polarized
double bound is commonly called the "Michael acceptor." As noted
above, the polarized double bond generated from the beta
elimination reaction described in reference to block 14 may serve
as a Michael acceptor. A fluorescent reporter having a nucleophilic
terminal group may serve as a Michael donor and react with the
polarized double bond of the fluorescent particle to couple the
fluorescent reporter to the fluorescent particle via the
nucleophilic terminal group. As with the phosphorylation and beta
elimination steps described above in reference to blocks 12, the
process of coupling the fluorescent reporter to the fluorescent
particle may, in some embodiments, be conducted in the presence of
an ionic liquid and, in some cases, an ionic liquid heated by
microwaves. The inclusion of the ionic liquid may desirably reduce
the generation of byproducts during the coupling process,
increasing the yield of the ensuing particle composite. In
addition, microwave heating may increase the rate of reaction for
the coupling process, reducing production time.
[0026] In general, the fluorescent reporter may include a compound
of any photoluminescent material (e.g., fluorophores, fluorescent
dyes, or other fluorescent materials) with a nucleophilic terminal
group coupled thereto. In addition, the nucleophilic terminal group
may include any nucleophile, such as but not limited to thiol and
amino groups. In some embodiments, it may be desirable to employ
hydrophilic fluorescent compounds within the fluorescent reporter
to insure a strong fluorescence signal may be subsequently
measured. In particular, assays are typically conducted in an
aqueous medium. If a hydrophobic dye is used in an aqueous medium,
it gets quenched and fluorescence signal is affected. The presence
of thiol may, in some embodiments, impart hydrophilicity and,
therefore, may be preferred as a nucleophilic terminal group of a
fluorescence reporter in some cases. Although there is some
ambiguity in the categorizations of hydrophilic compounds and
hydrophobic compounds in the chemical arts, the reference of
hydrophilic compounds as used herein may specifically refer to
compounds which do not dissolve in an organic solvent (e.g., ethyl
acetate).
[0027] In addition or alternative to being hydrophilic, the
fluorescent compound of the fluorescent reporter may be configured
to emit fluorescence within a different wavelength range than the
one or more fluorescent materials within the support substrate of
the fluorescent particle. In this manner, the fluorescence
emissions from the different fluorescent materials within the
support structure and the fluorescence reporter may not overlap.
Such a feature may be particularly advantageous for embodiments in
which the fluorescent materials within the support structure are
used to categorized particles within a sample. More specifically, a
variation of fluorescence ranges among the fluorescent materials
may allow different particle categorizations within a sample to be
detected without interfering with the detection of kinase activity
within the sample. In some cases, the fluorescent compound of the
fluorescent reporter may be configured to emit fluorescence within
a wavelength range greater than approximately 500 nm since many
assays include molecules having intrinsic fluorescence at
wavelengths less than approximately 450 nm. In some embodiments, an
assay may not be configured with such generalities and, therefore,
the configuration of the fluorescent reporter's fluorescence
emissions may vary among different applications. For example, in
some embodiments, the fluorescent compound of the fluorescent
reporter may be configured to emit fluorescence within a wavelength
range between approximately 400 nm and approximately 580 nm.
[0028] In some cases, the fluorescent reporter may include one or
more spacer compounds interposed between the nucleophilic terminal
group and the fluorescent compound. The inclusion of one or more
spacer compounds may lessen the interference of kinase activity
detection and the recognition process between the enzyme and the
substrate. In some embodiments, spacer compounds which collectively
include between approximately 1 atom and approximately 25 atoms may
be used to provide sufficient spacing between the nucleophilic
terminal group and the fluorescent compound. In more specific
cases, spacer compounds which collectively include between
approximately 5 atoms and approximately 25 atoms may be used. It is
noted that spacer compounds which collectively include greater than
25 atoms may be used in some applications, depending on the
specifications of the fluorescent particle and/or fluorescent
reporter. Examples of some spacer compounds having nucleophilic
terminal groups that may be employed with fluorescence compounds
for the fluorescence reporters are noted below. It is noted that
such a listing is exemplary and does not exclude the consideration
of other compounds for the fluorescence reporters described herein.
Other examples of spacer compounds are noted below in the listing
of exemplary fluorescent reporters that may be used for the methods
described herein.
Examples of Michael Donors
##STR00001##
[0030] Examples of some fluorescence reporters which may be used
for the methods described herein are noted below. It is noted that
such a listing is exemplary and does not exclude the consideration
of other fluorescence reporters which may be used for the methods
described herein.
Examples of Fluorescence Reporters
##STR00002##
[0032] An exemplary scheme performing the method described in FIG.
1 is outlined below. It is noted that such a scheme is exemplary
and does not exclude the consideration of other schemes which may
be used for processing a fluorescent particle for kinase detection.
Therefore, the methods described herein are not necessarily
restricted to the scheme outlined below. As shown below, a
kinase-specific peptide substrate may be coupled to a fluorescent
particle via a carboxylic acid functional group of the fluorescent
particle. As noted above in reference to block 10 in FIG. 1, the
methods described herein may be applied to fluorescent particles
having other functional groups. The coupling process may be
performed in the presence of ethyl-dimethylaminopropyl-carbodiimide
(EDC) and sulfonated n-hydroxysuccinimide, but other reagents may
be used. Subsequent to coupling the kinase-specific peptide
substrate to the fluorescent particle, a phosphorylation process
may be performed on the peptide substrate. As noted in the outlined
scheme, the phosphorylation process may be performed in the
presence of a kinase and ATP and, in some cases, an ionic liquid.
After a phosphate group is added to the peptide substrate, the
process continues to a beta-elimination step in which the peptide
substrate is dephosphorylated and a polarized double bond is
generated at a dephosphorylated site of the peptide substrate. As
noted above, such a beta-elimination step may be base-catalyzed
and, in some embodiments, may additionally be performed in the
presence of an ionic liquid. As noted in the scheme outlined below,
the polarized double bond may carry an amino acid-serine. The
methods described herein, however, may be configured to generate
other amino acid residues, such as amino acid threonine and
tyrosine.
[0033] Subsequent to the beta-elimination process, a fluorescent
reporter is adhered to the fluorescent particle via the polarized
double bond. The scheme below outlines two exemplary Michael
reactions for coupling fluorescence reporters to the fluorescent
particle. One of the Michael reactions includes coupling a
fluorescent reporter including a fluorescent compound (referenced
as a "reporter dye") bound through an amide bond (CONH bond) to one
or more spacer compounds (references as "spacer"). The CONH bond is
the result of coupling the fluorescent compound with the one or
more spacer compounds, specifically coupling a carboxylic acid
functional group of the fluorescent compound to an amino acid
functional group of the one or more spacer compounds. The inclusion
of the CONH bond is exemplary and, therefore, the methods described
herein are not necessarily so restricted. The other Michael
reaction depicted in the scheme outlined below includes coupling an
exemplary biotin-substituted Michael donor and a Streptavidin
conjugated fluorescent compound (referenced as a "reporter dye") to
the fluorescent particle. An exemplary fluorescent compound which
may be particularly suitable for such a reaction is Phycoerythrin
(PE). However, other fluorescent compounds as well as other
biotin-substituted Michael donors may be used for the methods
described herein.
##STR00003##
[0034] Due to the method outlined in FIG. 1, a number of
fluorescent particles are described herein. In particular, a number
of different fluorescent particle configurations are provided as a
result of each of the processing steps of the method. For example,
a particle is provided which includes a support substrate with one
or more fluorescent materials and a peptide substrate coupled to
the support substrate via a functional group of the support
substrate. In some embodiments, the peptide substrate may be
configured for specific kinase activity, such as for the particle
described in reference to block 10 of FIG. 1. In other cases, the
peptide substrate may be a phosphorylated peptide substrate,
resulting from the phosphorylation step described in reference to
block 12 of FIG. 1. In yet other cases, the peptide substrate may
include a Michael acceptor as a result of the processing step
described in reference to block 14 in FIG. 1. Yet further, the
fluorescent particle may include a fluorescent reporter coupled to
the peptide substrate as a result of the processing step described
in reference to block 16 in FIG. 1. The fluorescent reporter may
include any of the embodiments described above in reference to
block 16 and, for the sake of brevity, are not reiterated here.
[0035] In addition to the method described in reference to FIG. 1
and the resulting particles, various kits for performing the method
described in reference to FIG. 1 are provided. In particular, a kit
is provided which includes a plurality of fluorescent particles and
one or more kinase-specific peptide substrates. Each of the
plurality of fluorescent particles includes a support substrate
with one or more fluorescent materials configured to emit
fluorescence in a first wavelength range. In some cases, the one or
more kinase-specific peptide substrates are coupled to the support
substrates via functional groups of the support substrates. In
specific embodiments, the one or more kinase-specific peptide
substrates may be respectively coupled to different subsets of the
plurality of fluorescent particles. In this manner, a number of
different kinase activities may be evaluated within a sample and,
thus, determination of kinase activity may be multiplexed. In other
cases, however, the one or more kinase-specific peptide substrates
are isolated from the plurality of fluorescent particles. In such
embodiments, the kit may, in some cases, further include a reagent
configured to couple the one or more kinase-specific peptide
substrates to functional groups of the support substrates. In this
manner, the kit may be configured to produce particles with the
features described in reference to block 10 in FIG. 1.
[0036] In some cases, the kit may also include a phosphorylation
reagent configured to phosphorylate the one or more kinase-specific
peptide substrates to produce particles with the features described
in reference to block 12 in FIG. 1. Moreover, the kit may include
beta-elimination reagent configured to dephosphorylate the one or
more kinase-specific peptide substrates and generate Michael
acceptors at the dephosphorylation sites of the one or more
kinase-specific peptide substrates to produce particles with the
features described in reference to block 14 in FIG. 1. Further yet,
the kit may include one or more fluorescent reporter reagents each
having a nucleophilic terminal group and one or more fluorescent
compounds to produce particles with the features described in
reference to block 16 in FIG. 1. The fluorescent compounds of the
fluorescent reporter reagents may include any of the embodiments
described above in reference to block 16 and, for the sake of
brevity, are not reiterated here. In addition to the aforementioned
components, the kit described herein may be configured such that at
least one of the phosphorylation reagent, the beta-elimination
reagent, and the one or more fluorescent reporter reagents includes
an ionic liquid. Alternatively stated, the kit may be configured
such that any one or multiple of the reagents may be used in the
presence or the absence of an ionic liquid. In any case, the kit
may contain other ingredients which are considered standard for
assay preparation, such as but not limited to a fluorescent label,
a competitor molecule, a reference material, wash buffer,
plasticware, etc.
[0037] As noted above, a method for determining kinase activity
within a sample using a multiplexing assay scheme is outlined in a
flowchart in FIG. 2. As shown in FIG. 2, the method may include
block 20 in which a pooled population of different subsets of
fluorescent particles are exposed to a sample. The sample may
include any biological or chemical assay in which multiple analytes
are desired to be analyzed. At least some of the fluorescent
particles within the pooled population include a support substrate
with one or more fluorescent materials configured to emit
fluorescence in a first wavelength range and a peptide substrate
coupled to the support substrate via a functional group of the
support substrate. Such particles may be similar to the fluorescent
particle described in reference to block 10 of FIG. 1.
[0038] To facilitate a multiplexing scheme in which multiple
analytes within the sample are detected and/or quantified; the
fluorescent particles are configured into distinguishable groups.
In some cases, the groups are differentiated by different types
and/or concentrations of fluorescent materials absorbed into
particles and/or bound to the surface of particles. Consequently,
in some embodiments, at least some of the different subsets of
fluorescent particles within the pooled population may respectively
include a different configuration of the one or more fluorescent
materials. In one example, employing two dyes at 10 different
concentrations among a set of particles produces 100 fluorescently
distinguishable particle categories. The number of particle
categories may be augmented by increasing the number of dyes and/or
different dye intensities. In many cases, it is advantageous to
configure an assay for analysis for several analytes, such as on
the order of 100 or more different analytes so that time and
processing costs may be minimized in evaluating a sample. It is
noted that particle classifications may be facilitated in other
manners, such as by particle size and, consequently, the method
described in reference to FIG. 2 is not necessarily limited to
having subsets of particles with different configurations of
fluorescent materials. In addition to having differentiating
classification parameters, at least some of the different subsets
of fluorescent particles may, in some cases, respectively include a
different peptide substrate. In this manner, a number of different
kinase activities may be evaluated within the sample and, thus,
determination of kinase activity may be multiplexed along with the
identification of particles within the sample.
[0039] Continuing to block 22, the method includes exposing the
sample and the pooled population to a phosphorylation process that
is configured to add phosphate groups to accepting residues of the
peptide substrates (e.g., serine, threonine or tyrosine residues).
Such a process may be similar to the phosphorlating process
described in reference to block 12 in FIG. 1 and, therefore, is
referenced for the sake of brevity. Block 22 differs slightly from
the process described in block 12 in that the sample and pooled
population are exposed to a phosphorylation process. The matter of
whether the peptide substrate is phosphorylated depends on the
nature of the sample to inhibit or promote kinase activity. In
particular, it is noted that in some embodiments the peptide may
not be phosphorylated by the process described in reference to
block 22 due to kinase inhibitors within the sample. In such cases,
the method described in FIG. 2 may be used to determine negligible
or no kinase activity within the sample. Conversely, in embodiments
in which kinase activity is not inhibited (or even promoted) within
a sample, the method described in FIG. 2 may be used to determine
the degree of kinase activity. As such, although the processing
steps of the method outlined in FIG. 2 are configured to process a
fluorescent particle in a similar manner as described in FIG. 1,
the method in FIG. 2 is slightly different in that the particles
may not be altered by the processing steps. Rather the method in
FIG. 2 merely exposes the particles to processing steps configured
to alter the particles in the event kinase activity is present. In
this manner, the method may be used to determine a degree of kinase
activity within the sample including embodiments in which no or
negligible activity is present. A similar differentiation between
blocks 24 and 26 of FIG. 2 (which are described in more detail
below) and blocks 14 an 16 may be made as well.
[0040] As shown in FIG. 2, the method for determining kinase
activity within a sample continues to block 24 in which a plurality
of the fluorescent particles are processed such that if any
phosphorylated peptide substrates exist among the plurality of
fluorescent particles, the phosphorylated peptide substrates are
dephosphorylated and polarized double bonds are generated at
dephosphorylated sites of the peptide substrates. Such a process
may be similar to the process described in reference to block 14 in
FIG. 1 and, therefore, is referenced for the sake of brevity. In
addition, the method may include block 26 for further processing
the plurality of the fluorescent particles such that if any
polarized double bonds exist among the dephosphorylated sites of
the peptide substrates, fluorescent reporters are coupled to the
fluorescent particles at positions of the polarized double bonds
via nucleophilic terminal groups of the fluorescent reporters. Such
a process may be similar to the process described in reference to
block 16 in FIG. 1 and, therefore, is referenced for the sake of
brevity. In addition, the description of different embodiments of
fluorescent reporters described for block 16 may be referenced for
block 26 as well. In some cases, block 26 may include coupling
different fluorescent reporters to different subsets of the
fluorescent particles. In such cases, the different fluorescent
reporters may be configured to emit fluorescence in different
wavelength ranges (i.e., distinct from each other as well as the
wavelength range of the fluorescence material included in the
support structures of the fluorescent particles. In this manner,
the different fluorescent reporters may be configured to detect
different kinase activity and, therefore, kinase activity detection
may be multiplexed in the assay.
[0041] A further step of the method includes block 28 in which
fluorescence emissions of the plurality of the fluorescent
particles are measured. Such a measurement process may be performed
by any known measurement process, such as by flow cytometry or
fluorescence imaging. The method further includes block 30 in which
particle categorizations are determined based upon measured
fluorescence emissions within a first wavelength range as specified
by the fluorescent materials included with the support structures
of the fluorescent particles. More specifically, the output signals
generated from fluorescence emitted by the fluorescent materials
within the support structures of the particles may be used to
categorize the particles into different classification groups. In
addition thereto, the method includes determining, based upon the
existence of or lack of measured fluorescence emissions within the
second wavelength range, an amount of kinase activity within the
sample when the sample and the pooled population are exposed to the
phosphorylation process as shown by block 32. In particular, since
the presence of kinase activity is indicated by the phosphorylation
of a peptide substrate and the method described herein recognizes
such activity by placing a fluorescent reporter at sites which were
previously phosphorylated, fluorescence emissions from the
fluorescent reporter may be indicative of a degree of kinase
activity within a sample. Conversely, in embodiments in which
kinase activity is inhibited within a sample, phosphorylation of a
peptide substrate on a fluorescent particle will not occur nor will
coupling a fluorescent reporter to a fluorescent particle occur. As
a result, no fluorescence emissions will be detected for the
wavelength range of the fluorescence reporter, indicating
negligible or no kinase activity within the sample.
[0042] Further modifications and alternative embodiments of various
aspects of the invention may be apparent to those skilled in the
art in view of this description. For example, although the methods,
particles, and kits are particularly described for determining
kinase activity in a sample of a multiplexing scheme, the methods,
particles, and kits may alternatively be used for kinase detection
in a singleplexing scheme. Accordingly, this description is to be
construed as illustrative only and is for the purpose of teaching
those skilled in the art the general manner of carrying out the
invention. It is to be understood that the forms of the invention
shown and described herein are to be taken as the presently
preferred embodiments. Elements and materials may be substituted
for those illustrated and described herein, parts and processes may
be reversed, and certain features of the invention may be utilized
independently, all as would be apparent to one skilled in the art
after having the benefit of this description of the invention.
Changes may be made in the elements described herein without
departing from the spirit and scope of the invention as described
in the following claims.
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